Genetic modification in the gene for human G protein beta3 subunit for the diagnosis of diseases

ABSTRACT

The present invention relates to the use of a mutation in the gene for human G-protein β3 sub-unit for diagnosing illnesses.

[0001] The use of a genetic modification in the gene for human G proteinβ3 subunit for the diagnosis of diseases

[0002] The present invention relates to a method for the diagnosis ofdiseases by genetic analysis, in particular the analysis of genes forsubunits of the human guanine nucleotide-binding proteins (G proteins).

[0003] Heterotrimeric guanine nucleotide-binding proteins (G proteins)have an outstanding importance in intracellular signal transduction.They mediate the relaying of extracellular signals after stimulation ofhormone receptors and other receptors which undergo a conformationalchange after receptor activation. This leads to activation of G proteinswhich may subsequently activate or inhibit intracellular effectors (eg.ion channels, enzymes). Heterotrimeric G proteins consist of threesubunits, the α, β and γ subunits. To date, several different asubunits, 5βsubunits and about 12γ subunits have been detected bybiochemical and molecular biological methods (Birnbaumer, L. andBirnbaumer, M. Signal transduction by G proteins: 1994 edition.J.Recept.Res. 15:213-252, 1995; Offermanns, S. and Schultz, G. Complexinformation processing by the transmembrane signaling system involving Gproteins. Naunyn Schmiedebergs Arch.Pharmacol. 350:329-338, 1994;Nürnberg, B., Gudermann, T., and Schultz, G. Receptors and G proteins asprimary components of transmembrane signal transduction. Part 2. Gproteins: structure and function. J.Mol.Med. 73:123-132, 1995; Neer, E.J. Heterotrimeric G proteins: Organizers of Transmembrane Signals. Cell80:249-257, 1995; Rens-Domiano, S. and Hamm, H. E. Structural andfunctional relationships of heterotrimeric G-proteins. FASEB J.9:1059-1066, 1995).

[0004] Receptor-mediated activation of certain a subunits can beinhibited by pretreatment with pertussis toxin (?TX). These include, inparticular, the α isoforms αi1, αi2 and αi3, and various α subunits. Gproteins of these types are also referred to as PTX-sensitive Gproteins.

[0005] We have found that a genetic modification in the gene for human Gprotein β3 subunits is suitable for the diagnosis of diseases. Thisgenetic modification is particularly suitable for establishing the riskof developing a disorder associated with G protein dysregulation.

[0006] The invention furthermore relates to a method for establishing arelative risk of developing disorders associated with G proteindysregulation for a subject, which comprises comparing the gene sequencefor human G protein β3 subunit of the subject with the gene sequence SEQID NO:1, and, in the event that a thymine (T) is present at position825, assigning the subject an increased risk of disease.

[0007] The genetic modification which has been found is located in thegene for human G protein β3 subunit. This gene has been described byLevine et al. (Proc. Natl. Acad. Sci USA, 87, (1990) 2329-2333). Thecoding region has an Ser codon (TCC) at position 275, while subjectswith an increased risk of a disease associated with G proteindysregulation have the codon TCT, which likewise codes for Ser, at thisposition. The genetic modification is a base substitution at position825 in which a cytosine (C) is replaced by thymine (T). However, thisbase exchange is “silent” at the amino-acid level, .e. it does not leadto incorporation of a different amino acid at this position. Thesequence found in subjects with an increased risk of disease is depictedin SEQ ID NO:1 in the sequence listing.

[0008] The genetic modification which has been found usually occurs inheterozygous form.

[0009] Disorders associated with G protein dysregulation are defined asdiseases in which the G protein is involved in signal transduction anddoes not carry out its function in a physiological manner.

[0010] The dysregulation may have a number of causes, for example amodification in the structural gene or modified gene expression.

[0011] The disorders include cardiovascular diseases, metabolicdisturbances and immunological diseases.

[0012] Cardiovascular diseases which may be mentioned are:

[0013] Hypertension, pregnancy hypertension (gestosis, hypertension inpregnancy), coronary heart disease, localized and/or generalizedatherosclerosis, stenoses of blood vessels, restenosis afterrevascularizing procedures (eg. PTCA with and without stentimplantation), tendency to stroke or thrombosis and increased plateletaggregation.

[0014] Metabolic disturbances which may be mentioned are:

[0015] Metabolic syndrome, insulin resistance and hyperinsulinemia, typeII diabetes rellitus, diabetic complications (eg. nephropathy,neuropathy, retinopathy, etc.) disturbances of lipid metabolism,disturbances of central chemoreception (CO₂ tolerance, acidosistolerance, sudden infant death (SIDS)).

[0016] Immunological diseases which may be mentioned are:

[0017] Impaired strength of the body's immune response (formation ofimmunoglobulins, aggressiveness of T cells and NK cells), impairedgeneral tendency to proliferation, including wound-healing capacity,tendency to develop tumors and proliferation including metastasizingpotential of malignantly transformed cells, duration of the latencyperiod after HIV infection until the disease becomes clinically evident,Kaposi sarcoma, tendency to cirrhosis of the liver, transplant toleranceand transplant rejection.

[0018] The use of the genetic mutation according to the invention isparticularly suitable for establishing the risk of developinghypertension.

[0019] The invention furthermore relates to the production of transgenicanimals harboring the genetic mutation described above. Transgenicanimals of this type are of great importance in particular as animalmodels for the investigation and therapy of the disorders describedabove. The methods for generating transgenic animals are generally knownto the skilled worker.

[0020] For the method according to the invention for establishing therelative risk of developing a disease, body material containing thesubject's genetic information is taken from a subject. This is achievedas a rule by taking blood and isolating .he nucleic acid therefrom.

[0021] The structure of the gene for the G protein β3 subunit isestablished from the subject's isolated nucleic acid and is comparedwith the sequence indicated in SEQ ID NO:1.

[0022] The structure of the gene can be established by sequencing of thenucleic acid. This can take place either directly from the genomic DNAor after amplification of the nucleic acid, for example by the PCRtechnique.

[0023] The structure of the gene can take place at the genomic level orelse at the mRNA or cDNA level.

[0024] It is preferably established by sequencing after PCRamplification of the cDNA. The primers suitable for the PCR can easilybe inferred by the skilled worker from the sequences depicted in SEQ IDNO:1. The procedure for this is advantageously such that in each case aprimer binding a strand and complementary strand in front of and behindthe relevant base position 825 is chosen.

[0025] However, other methods can also be used for comparison of thegenes, for example selective hybridization or appropriate mapping withrestriction enzymes. The C→T base exchange at the position 825 describedabove leads to loss of a cleavage site for the restriction enzyme Dsa I,which is likewise used to detect this genetic polymorphism.

[0026] If the subject has a thymine (T) at position 825, he is to beassigned a greater risk of disease than a subject with a cytosine (C) atthis position.

[0027] The invention is illustrated further in the following examples.

EXAMPLE 1

[0028] Detection of the Genetic Modification in Hypertensives bySequencing

[0029] An enhanced susceptibility to activation of PTX-sensitive Gproteins was detected in preliminary investigations on patients withessential hypertension. This detection was possible in immortalizedcells from patients having as phenotypical marker an enhanced activityof the Na/H exchanger. The enhanced susceptibility to activation ofPTX-sensitive G proteins has important consequences for cellularfunction. These include enhanced formation of intracellular secondmessenger molecules (eg. inositol 1,4,5-trisphosphate), enhanced releaseof intracellular Ca²⁺ ions, increased formation of immunoglobulins andan increased rate of cell growth. Since these changes can be detected inimmortalized cells and after a long duration of cell culturing, it maybe assumed that this modification is genetically fixed (Rosskopf, D.,Frömter, E., and Siffert, W. Hypertersive sodium-proton exchangerphenotype persists in immortalized lymphoblasts from essentialhypertensive patients—a cell culture model for human hypertension.J.Clin.Invest. 92:2553-2559, 1993; Rosskopf, D., Hartung, K., Rense, J.,and Siffert, W. Enhanced immunoglobulin formation of immortalized Bcells from hypertensive patients. Hypertension 26:432-435, 1995;Rosskopf, D., Schröder, K. -J., and Siffert, e. Role of sodium-hydrogenexchange in the proliferation of immortalised lymphoblasts from patientswith essential hypertension and normotensive subjects. Cardiovasc.Res.29:254-259, 1995; Siffert, W., Rosskopf, D., Moritz, A., Wieland, T.,Kaldenberg-Stasch, S., Kettler, N., Hartung, K., Beckmann, S., andJakobs, K. H. Enhanced G protein activation in immortalized lymphoblastsfrom patients with essential hypertension. J.Clin.Invest. 96:759-766,1995).

[0030] RNA was prepared by standard methods from immortalized cell linesfrom hypertensives and was transcribed into cDNA using reversetranscriptase. Using the polymerase chain reaction (PCR), the cDNAcoding for the G protein β3 subunit was amplified and sequenced. Thefollowing oligonucleotide primers were employed for the PCR: 5′-TGG GGGAGA TGG AGC AAC TG and 5′-CTG CTG AGT GTG TTC ACT GCC.

[0031] Compared with the sequence published by Levine et al. (Levine, M.A., Smallwood, P. M., Moen, P. T., Jr., Helman, L. J., and Ahn, T. G.Molecular cloning of β3 subunit, a third form of the G protein β-subunitpolypeptide. Proc. Natl. Acad. Sci. USA 87(6):2329-2333, 1990), thefollowing difference was found in the cDNA from hypertensives' cells:nucleotide 825 cytosine (C) in the region of the coding sequence isreplaced by a thymine (T) (nuc-leotide 1 corresponds to base A in theATG start codon). This base exchange leads to a silent polymorphism, ie.the amino acid encoded by the corresponding base triplet (serine) is notaltered by comparison with the original sequence. The DNA sequence foundis described in SEQ ID NO:1.

EXAMPLE 2

[0032] Detection of the Genetic Modification in Hypertensives byRestriction Enzyme Analysis

[0033] The figure depicts a comparison of genes from normotensives andhypertensives by restriction enzyme analysis. In this, the cDNA codingfor β3 from cells from normotensives (NT) and hypertensives (RT), whichhad been amplified by PCR, was subjected to a restriction enzymeanalysis using the enzyme Dsa I. The reaction products were fractionatedin an agarose gel, which is depicted in the figure.

[0034] The complete restriction of β3 cDNA from normotensive cells afterdigestion with Dsa I is nearly evident from the figure. The cDNA fromhypertensives' cells is only partly cut by Dsa I. Apart from thecleavage products to be expected there is also uncleaved PCR product.Reference fragments (markers) are loaded on the left and right forcomparison of sizes. Four of the five DNA sequences from hypertensivesdepicted here show the base exchange described above and areheterozygous for this modification.

1 4 1 1517 DNA Homo sapiens 1 atgggggaga tggagcaact gcgtcaggaagcggagcagc tcaagaagca gattgcagat 60 gccaggaaag cctgtgctga cgttactctggcagagctgg tgtctggcct agaggtggtg 120 ggacgagtcc agatgcggac gcggcggacgttaaggggac acctggccaa gatttacgcc 180 atgcactggg ccactgattc taagctgctggtaagtgcct cgcaagatgg gaagctgatc 240 gtgtgggaca gctacaccac caacaaggtgcacgccatcc cactgcgctc ctcctgggtc 300 atgacctgtg cctatgcccc atcagggaactttgtggcat gtggggggct ggacaacatg 360 tgttccatct acaacctcaa atcccgtgagggcaatgtca aggtcagccg ggagctttct 420 gctcacacag gttatctctc ctgctgccgcttcctggatg acaacaatat tgtgaccagc 480 tcgggggaca ccacgtgtgc cttgtgggacattgagactg ggcagcagaa gactgtattt 540 gtgggacaca cgggtgactg catgagcctggctgtgtctc ctgacttcaa tctcttcatt 600 tcgggggcct gtgatgccag tgccaagctctgggatgtgc gagaggggac ctgccgtcag 660 actttcactg gccacgagtc ggacatcaacgccatctgtt tcttccccaa tggagaggcc 720 atctgcacgg gctcggatga cgcttcctgccgcttgtttg acctgcgggc agaccaggag 780 ctgatctgct tctcccacga gagcatcatctgcggcatca cgtctgtggc cttctccctc 840 agtggccgcc tactattcgc tggctacgacgacttcaact gcaatgtctg ggactccatg 900 aagtctgagc gtgtgggcat cctctctggccacgataaca gggtgagctg cctgggagtc 960 acagctgacg ggatggctgt ggccacaggttcctgggaca gcttcctcaa aatctggaac 1020 tgaggaggct ggagaaaggg aagtggaaggcagtgaacac actcagcagc cccctgcccg 1080 accccatctc attcaggtgt tctcttctatattccgggtg ccattcccac taagctttct 1140 cctttgaggg cagtggggag catgggactgtgcctttggg aggcagcatc agggacacag 1200 gggcaaagaa ctgccccatc tcctcccatggccttccctc cccacagtcc tcacagcctc 1260 tcccttaatg agcaaggaca acctgcccctccccagccct ttgcaggccc agcagacttg 1320 agtctgaggc cccaggccct aggattcctcccccagagcc actacctttg tccaggcctg 1380 ggtggtatag ggcgtttggc cctgtgactatggctctggc accactaggg tcctggccct 1440 cttcttattc atgctttctc ctttttctacctttttttct ctcctaagac acctgcaata 1500 aagtgtagca ccctggt 1517 2 340 PRTHomo sapiens 2 Met Gly Glu Met Glu Gln Leu Arg Gln Glu Ala Glu Gln LeuLys Lys 1 5 10 15 Gln Ile Ala Asp Ala Arg Lys Ala Cys Ala Asp Val ThrLeu Ala Glu 20 25 30 Leu Val Ser Gly Leu Glu Val Val Gly Arg Val Gln MetArg Thr Arg 35 40 45 Arg Thr Leu Arg Gly His Leu Ala Lys Ile Tyr Ala MetHis Trp Ala 50 55 60 Thr Asp Ser Lys Leu Leu Val Ser Ala Ser Gln Asp GlyLys Leu Ile 65 70 75 80 Val Trp Asp Ser Tyr Thr Thr Asn Lys Val His AlaIle Pro Leu Arg 85 90 95 Ser Ser Trp Val Met Thr Cys Ala Tyr Ala Pro SerGly Asn Phe Val 100 105 110 Ala Cys Gly Gly Leu Asp Asn Met Cys Ser IleTyr Asn Leu Lys Ser 115 120 125 Arg Glu Gly Asn Val Lys Val Ser Arg GluLeu Ser Ala His Thr Gly 130 135 140 Tyr Leu Ser Cys Cys Arg Phe Leu AspAsp Asn Asn Ile Val Thr Ser 145 150 155 160 Ser Gly Asp Thr Thr Cys AlaLeu Trp Asp Ile Glu Thr Gly Gln Gln 165 170 175 Lys Thr Val Phe Val GlyHis Thr Gly Asp Cys Met Ser Leu Ala Val 180 185 190 Ser Pro Asp Phe AsnLeu Phe Ile Ser Gly Ala Cys Asp Ala Ser Ala 195 200 205 Lys Leu Trp AspVal Arg Glu Gly Thr Cys Arg Gln Thr Phe Thr Gly 210 215 220 His Glu SerAsp Ile Asn Ala Ile Cys Phe Phe Pro Asn Gly Glu Ala 225 230 235 240 IleCys Thr Gly Ser Asp Asp Ala Ser Cys Arg Leu Phe Asp Leu Arg 245 250 255Ala Asp Gln Glu Leu Ile Cys Phe Ser His Glu Ser Ile Ile Cys Gly 260 265270 Ile Thr Ser Val Ala Phe Ser Leu Ser Gly Arg Leu Leu Phe Ala Gly 275280 285 Tyr Asp Asp Phe Asn Cys Asn Val Trp Asp Ser Met Lys Ser Glu Arg290 295 300 Val Gly Ile Leu Ser Gly His Asp Asn Arg Val Ser Cys Leu GlyVal 305 310 315 320 Thr Ala Asp Gly Met Ala Val Ala Thr Gly Ser Trp AspSer Phe Leu 325 330 335 Lys Ile Trp Asn 340 3 20 DNA Homo sapiens 3tgggggagat ggagcaactg 20 4 21 DNA Homo sapiens 4 ctgctgagtg tgttcactgc c21

We claim:
 1. The use of a genetic modification in the gene for human Gprotein β3 subunit for the diagnosis of diseases.
 2. The use of agenetic modification in the gene for human G protein β3 subunit forestablishing the risk of developing a disorder associated with G proteindysregulation.
 3. The use as claimed in claim 2, wherein the geneticmodification is in the codon for amino acid 275 in SEQ ID NO:1.
 4. Theuse as claimed in claim 3, wherein there is substitution of cytosine bythymine in position 825 in SEQ ID NO:1.
 5. The use as claimed in claim2, wherein the disorder is a cardiovascular disease, a metabolicdisturbance or an immunological disease.
 6. The use as claimed in claim2, wherein the disorder is hypertension.
 7. A method for establishing arelative risk of developing disorders associated with G proteindysregulation for a subject, which comprises comparing the gene sequencefor human C protein β3 subunit of the subject with the gene sequence SEQID NO:1, and, in the event that a thymine (T) is present at position825, assigning the subject an increased risk of disease.
 8. A method asclaimed in claim 7, wherein the comparison of genes is carried out bysequencing.
 9. A method as claimed in claim 8, wherein a gene sectionwhich includes position 825 is amplified before the sequencing.
 10. Amethod as claimed in claim 7, wherein the comparison of genes is carriedout by hybridization.
 11. A method as claimed in claim 7, wherein thecomparison of genes is tarried out by cleavage using restrictionenzymes.
 12. A method as claimed in claim 11, wherein the restrictionenzyme Dsa I is used.